Effects of focused ultrasound in a "clean" mouse model of ultrasonic neuromodulation.

Recent studies on ultrasonic neuromodulation (UNM) in rodents have shown that focused ultrasound (FUS) can activate peripheral auditory pathways, leading to off-target and brain-wide excitation, which obscures the direct activation of the target area by FUS. To address this issue, we developed a new mouse model, the double transgenic Pou4f3+/DTR × Thy1-GCaMP6s, which allows for inducible deafening using diphtheria toxin and minimizes off-target effects of UNM while allowing effects on neural activity to be visualized with fluorescent calcium imaging. Using this model, we found that the auditory confounds caused by FUS can be significantly reduced or eliminated within a certain pressure range. At higher pressures, FUS can result in focal fluorescence dips at the target, elicit non-auditory sensory confounds, and damage tissue, leading to spreading depolarization. Under the acoustic conditions we tested, we did not observe direct calcium responses in the mouse cortex. Our findings provide a cleaner animal model for UNM and sonogenetics research, establish a parameter range within which off-target effects are confidently avoided, and reveal the non-auditory side effects of higher-pressure stimulation.

Effects of focused ultrasound in a "clean" mouse model of ultrasonic neuromodulation.

Recent studies on ultrasonic neuromodulation (UNM) in rodents have shown that focused ultrasound (FUS) can activate peripheral auditory pathways, leading to off-target and brain-wide excitation, which obscures the direct activation of the target area by FUS. To address this issue, we developed a new mouse model, the double transgenic Pou4f3+/DTR × Thy1-GCaMP6s, which allows for inducible deafening using diphtheria toxin and minimizes off-target effects of UNM while allowing effects on neural activity to be visualized with fluorescent calcium imaging. Using this model, we found that the auditory confounds caused by FUS can be significantly reduced or eliminated within a certain pressure range. At higher pressures, FUS can result in focal fluorescence dips at the target, elicit non-auditory sensory confounds, and damage tissue, leading to spreading depolarization. Under the acoustic conditions we tested, we did not observe direct calcium responses in the mouse cortex. Our findings provide a cleaner animal model for UNM and sonogenetics research, establish a parameter range within which off-target effects are confidently avoided, and reveal the non-auditory side effects of higher-pressure stimulation.

Focused ultrasound excites cortical neurons via mechanosensitive calcium accumulation and ion channel amplification.

Ultrasonic neuromodulation has the unique potential to provide non-invasive control of neural activity in deep brain regions with high spatial precision and without chemical or genetic modification. However, the biomolecular and cellular mechanisms by which focused ultrasound excites mammalian neurons have remained unclear, posing significant challenges for the use of this technology in research and potential clinical applications. Here, we show that focused ultrasound excites primary murine cortical neurons in culture through a primarily mechanical mechanism mediated by specific calcium-selective mechanosensitive ion channels. The activation of these channels results in a gradual build-up of calcium, which is amplified by calcium- and voltage-gated channels, generating a burst firing response. Cavitation, temperature changes, large-scale deformation, and synaptic transmission are not required for this excitation to occur. Pharmacological and genetic inhibition of specific ion channels leads to reduced responses to ultrasound, while over-expressing these channels results in stronger ultrasonic stimulation. These findings provide a mechanistic explanation for the effect of ultrasound on neurons to facilitate the further development of ultrasonic neuromodulation and sonogenetics as tools for neuroscience research.

Acoustically triggered mechanotherapy using genetically encoded gas vesicles.

Recent advances in molecular engineering and synthetic biology provide biomolecular and cell-based therapies with a high degree of molecular specificity, but limited spatiotemporal control. Here we show that biomolecules and cells can be engineered to deliver potent mechanical effects at specific locations inside the body through ultrasound-induced inertial cavitation. This capability is enabled by gas vesicles, a unique class of genetically encodable air-filled protein nanostructures. We show that low-frequency ultrasound can convert these biomolecules into micrometre-scale cavitating bubbles, unleashing strong local mechanical effects. This enables engineered gas vesicles to serve as remotely actuated cell-killing and tissue-disrupting agents, and allows genetically engineered cells to lyse, release molecular payloads and produce local mechanical damage on command. We demonstrate the capabilities of biomolecular inertial cavitation in vitro, in cellulo and in vivo, including in a mouse model of tumour-homing probiotic therapy.

Ultrasound Technologies for Imaging and Modulating Neural Activity.

Visualizing and perturbing neural activity on a brain-wide scale in model animals and humans is a major goal of neuroscience technology development. Established electrical and optical techniques typically break down at this scale due to inherent physical limitations. In contrast, ultrasound readily permeates the brain, and in some cases the skull, and interacts with tissue with a fundamental resolution on the order of 100 µm and 1 ms. This basic ability has motivated major efforts to harness ultrasound as a modality for large-scale brain imaging and modulation. These efforts have resulted in already-useful neuroscience tools, including high-resolution hemodynamic functional imaging, focused ultrasound neuromodulation, and local drug delivery. Furthermore, recent breakthroughs promise to connect ultrasound to neurons at the genetic level for biomolecular imaging and sonogenetic control. In this article, we review the state of the art and ongoing developments in ultrasonic neurotechnology, building from fundamental principles to current utility, open questions, and future potential.

Thermoplasmonic Optical Fiber for Localized Neural Stimulation.

Thermoplasmonic effect-based neural stimulation has been suggested as an alternative optical neural stimulation technology without genetic modification. Integration of near-infrared light with plasmonic gold nanoparticles has been demonstrated as a neuromodulation tool on in vitro neuronal network models. In order to further test the validity of the thermoplasmonic neural stimulation across multiple biological models (in vitro, ex vivo, and in vivo) avoiding genetic modification in optical neuromodulation, versatile engineering approaches to apply the thermoplasmonic effect would be required. In this work, we developed a gold nanorod attached optical fiber technology for the localized neural stimulation based on a thermoplasmonic effect. A simple fabrication process was developed for efficient nanoparticle coating on commercial optical fibers. The thermoplasmonic optical fiber proved that it can locally modulate the neural activity in vitro. Lastly, we simulated the spatiotemporal temperature change by the thermoplasmonic optical fiber and analyzed its applicability to in vivo animal models.

Single-Cell Photothermal Neuromodulation for Functional Mapping of Neural Networks.

Photothermal neuromodulation is one of the emerging technologies being developed for neuroscience studies because it can provide minimally invasive control of neural activity in the deep brain with submillimeter precision. However, single-cell modulation without genetic modification still remains a challenge, hindering its path to broad applications. Here, we introduce a nanoplasmonic approach to inhibit single-neural activity with high temporal resolution. Low-intensity near-infrared light was focused at the single cell size on a gold-nanorod-integrated microelectrode array platform, generating a photothermal effect underneath a target neuron for photothermal stimulation. We found that the photothermal stimulation modulates the spontaneous activity of a target neuron in an inhibitory manner. Single neuron inhibition was fast and highly reliable without thermal damage, and it can induce changes in network firing patterns, potentially suggesting their application for in vivo circuit modulation and functional connectomes.

Biomolecular Ultrasound and Sonogenetics.

Visualizing and modulating molecular and cellular processes occurring deep within living organisms is fundamental to our study of basic biology and disease. Currently, the most sophisticated tools available to dynamically monitor and control cellular events rely on light-responsive proteins, which are difficult to use outside of optically transparent model systems, cultured cells, or surgically accessed regions owing to strong scattering of light by biological tissue. In contrast, ultrasound is a widely used medical imaging and therapeutic modality that enables the observation and perturbation of internal anatomy and physiology but has historically had limited ability to monitor and control specific cellular processes. Recent advances are beginning to address this limitation through the development of biomolecular tools that allow ultrasound to connect directly to cellular functions such as gene expression. Driven by the discovery and engineering of new contrast agents, reporter genes, and bioswitches, the nascent field of biomolecular ultrasound carries a wave of exciting opportunities.

Electro-optical Neural Platform Integrated with Nanoplasmonic Inhibition Interface.

Engineering of neural interfaces with nanomaterials for remote manipulation facilitates the development of platforms for the study and treatment of brain disorders, yet extending their capability to inhibiting the electrical activities of unmodified neurons has been difficult. Here we report the development of an electro-optical neural platform integrated with gold nanorods for simultaneous electrical excitation and readout, and photothermal inhibition of neural activities. A monolayer of gold nanorods was placed at the electrode-neuron interfaces of a microelectrode array for photothermal stimulation of neural activities. This nanoplasmonic interface interacted well with neurons and metal electrodes without affecting the biological and electrical properties. We demonstrated that spontaneous firing of neurons and their signal propagation along the neurites evoked by electrical stimulation were optically inhibited on this neural platform. We believe that our platform could be an alternative to the optogenetic approach and may ultimately be applied to prosthetic devices based on optical neuromodulation.

Emerging neural stimulation technologies for bladder dysfunctions.

In the neural engineering field, physiological dysfunctions are approached by identifying the target nerves and providing artificial stimulation to restore the function. Neural stimulation and recording technologies play a central role in this approach, and various engineering devices and stimulation techniques have become available to the medical community. For bladder control problems, electrical stimulation has been used as one of the treatments, while only a few emerging neurotechnologies have been used to tackle these problems. In this review, we introduce some recent developments in neural stimulation technologies including microelectrode array, closed-loop neural stimulation, optical stimulation, and ultrasound stimulation.

Photothermal inhibition of neural activity with near-infrared-sensitive nanotransducers.

A neural stimulation technique that can inhibit neural activity reversibly and directly without genetic modification is valuable for understating complex brain functions and treating brain diseases. Here, we propose a near-infrared (NIR)-activatable nanoplasmonic technique that can inhibit the electrical activity of neurons by utilizing gold nanorods (GNRs) as photothermal transducers on cellular membranes. The GNRs were bound onto the plasma membrane of neurons and irradiated with NIR light to induce GNR-mediated photothermal heating near the membrane. The electrical activity from the cultured neuronal networks pretreated with GNRs was immediately inhibited upon NIR irradiation, and fully restored when NIR light was removed. The degree of inhibition could be precisely modulated by tuning the laser intensity, thereby enabling restoration of firing of a hyperactive neuronal network with epileptiform activity. This nanotechnological approach to inhibit neural activity provides a powerful therapeutic platform to control cellular functions associated with disordered neural circuits.

Generation of cellular micropatterns on a single-layered graphene film.

Micropatterns of fibroblast and hippocampal neurons are generated on a single-layered graphene substrate. A large-area (1 cm × 1 cm) graphene film on Si/SiO2 is functionalized by surface-initiated ATRP of non-biofouling oligo(ethylene glycol) methacrylate, after grafting of the polymerization initiator bearing α-bromoisobutylate via photoreaction of the perfluorophenyl azide group. The microcontact printing-assisted spatio-selective reaction, after chemical activation of the terminal hydroxyl group of oligo(ethylene glycol) in the polymeric film, is utilized to generate the patterns of fibroblast and hippocampal neurons.

Combined multisegmental surgical-orthodontic treatment of bialveolar protrusion and chin retrusion with severe facial asymmetry.

LeFort I osteotomy, anterior segmental osteotomy, bilateral sagittal split ramus osteotomy, and genioplasty are frequently used methods for correcting facial deformities. However, in patients with an abnormally shaped maxilla or mandible, more complex surgical techniques or multiple combinations must be considered for improved esthetic results. This article presents a patient with bialveolar protrusion, mandibular prognathism, chin retrusion, a long face, and severe facial asymmetry. A combination of LeFort I asymmetric impaction, anterior segmental osteotomy, and 3-piece segmentation of the maxilla, and bilateral sagittal split ramus osteotomy, anterior segmental osteotomy, genioplasty advancement, and angle shaving in the mandible were conducted simultaneously. In patients with complicated deformities that cannot be classified by simple conventional classification methods, multisegmental osteotomy can be an option for improved esthetic results.